Waveform-based classification of dentate spikes

Synchronous excitatory discharges from the entorhinal cortex (EC) to the dentate gyrus (DG) generate fast and prominent patterns in the hilar local field potential (LFP), called dentate spikes (DSs). As sharp-wave ripples in CA1, DSs are more likely to occur in quiet behavioral states, when memory consolidation is thought to take place. However, their functions in mnemonic processes are yet to be elucidated. The classification of DSs into types 1 or 2 is determined by their origin in the lateral or medial EC, as revealed by current source density (CSD) analysis, which requires recordings from linear probes with multiple electrodes spanning the DG layers. To allow the investigation of the functional role of each DS type in recordings obtained from single electrodes and tetrodes, which are abundant in the field, we developed an unsupervised method using Gaussian mixture models to classify such events based on their waveforms. Our classification approach achieved high accuracies (> 80%) when validated in 8 mice with DG laminar profiles. The average CSDs, waveforms, rates, and widths of the DS types obtained through our method closely resembled those derived from the CSD-based classification. As an example of application, we used the technique to analyze single-electrode LFPs from apolipoprotein (apo) E3 and apoE4 knock-in mice. We observed that the latter group, which is a model for Alzheimer’s disease, exhibited wider DSs of both types from a young age, with a larger effect size for DS type 2, likely reflecting early pathophysiological alterations in the EC-DG network, such as hyperactivity. In addition to the applicability of the method in expanding the study of DS types, our results show that their waveforms carry information about their origins, suggesting different underlying network dynamics and roles in memory processing.


Fig S4 .
Fig S4.Average waveform dynamics of each DS type of all mice.(A) Scaled mean waveforms of DS1 detected in the channel that yielded more DSs for all mice.(B) Same as A, but for DS2 (C) Second derivatives of the DS1 waveforms depicted in A. (D) Same as C, but for DS2.DS1: dentate spike type 1; DS2: dentate spike type 2.

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Fig S5.DS width based on waveform dynamics is more stable than width at half height.(A) The top panel shows the width for each DS type (Metric 4 -M4) across channels for each mouse.Colored bars correspond to the means, and the error bars indicate the standard deviations.The bottom panel shows the coefficient of variation of the corresponding measurements in the panel above.(B) Same as A, but for the width at half-height (Metric 5 -M5).(C) Coefficients of variation of the means of each metric across mice.(D) Mean coefficients of variation of each metric across mice.(E) Distributions of M4 and M5 of DS1 showing that M5 is more variable.(F) Same as E, but for DS2.CV: coefficient of variation; DS: dentate spike; DS1: dentate spike type 1; DS2: dentate spike type 2.

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Fig S8.DS waveform comparison between methods for each channel of mouse B2.The panels in the first and second columns show the waveforms of each DS type classified by CSDbC and WFbC respectively.The panels in the third and fourth columns compare the waveforms of DS1 and DS2 respectively.Each line represents the chosen channel for DS detection.CSD: current source density; CSDbC: CSD-based classification; DS: dentate spike; DS1: DS type 1; DS2: DS type 2; WFbC: waveform-based classification.

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Fig S9.DS waveform comparison between methods for each channel of mouse B3.The panels in the first and second columns show the waveforms of each DS type classified by CSDbC and WFbC respectively.The panels in the third and fourth columns compare the waveforms of DS1 and DS2 respectively.Each line represents the chosen channel for DS detection.CSD: current source density; CSDbC: CSD-based classification; DS: dentate spike; DS1: DS type 1; DS2: DS type 2; WFbC: waveform-based classification.

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Fig S10.DS waveform comparison between methods for each channel of mouse C1.The panels in the first and second columns show the waveforms of each DS type classified by CSDbC and WFbC respectively.The panels in the third and fourth columns compare the waveforms of DS1 and DS2 respectively.Each line represents the chosen channel for DS detection.CSD: current source density; CSDbC: CSD-based classification; DS: dentate spike; DS1: DS type 1; DS2: DS type 2; WFbC: waveform-based classification.

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Fig S11.DS waveform comparison between methods for each channel of mouse C2.The panels in the first and second columns show the waveforms of each DS type classified by CSDbC and

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Fig S14.DS-triggered CSDs from mice of dataset B. (A) Mean CSDs of mouse B1 within a 60-ms window around the peak of each DS type classified by CSDbC and WFbC.Blue and red colors represent sinks and sources, respectively.The estimated DG layers are indicated on the right.(B,C) Same as A, but for mice B2 and B3.CSD: current source density; CSDbC: CSD-based classification; DS: dentate spike; DS1: DS type 1; DS2: DS type 2; WFbC: waveform-based classification; gcl: granule cell layer; h: hilus; iml: inner molecular layer; mml: middle molecular layer; oml: outer molecular layer.

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Fig S15.DS-triggered CSDs from mice of dataset C. (A) Mean CSDs of mouse C1 within a 60-ms window around the peak of each DS type classified by CSDbC and WFbC.Blue and red colors represent sinks and sources, respectively.The estimated DG layers are indicated on the right.(B-D) Same as A, but for mice C2, C3 and C4.CSD: current source density; CSDbC: CSD-based classification; DS: dentate spike; DS1: DS type 1; DS2: DS type 2; WFbC: waveform-based classification; gcl: granule cell layer; h: hilus; iml: inner molecular layer; mml: middle molecular layer; oml: outer molecular layer.

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Fig S16.Misclassified DSs exhibit post-peak dynamics similar to related types.(A) The probability densities of DS1 misclassified as DS2 by WFbC in channels 7 to 11 of mouse A is shown on top.The bottom panel shows the same for DS2.(B) Mean CSDs for the well-classified (left panel) and misclassified (right panel) DS1 by WFbC.(C) Same as B, but for DS2.(D) Mean waveforms of well-and misclassified DSs of both types in channel 9. (E) Pair-to-pair comparison of the waveforms shown in D. The blue dots indicate when Cohen's D size effect is greater than 0.5 for periods with significant differences (p < 0.05 in ttest) between the waveforms.(F) Mean 2 nd derivatives of the waveforms shown in D. The dotted lines indicate the start and end width limits of each waveform.DS1: dentate spike type 1; DS2: dentate spike type 2; MC: misclassified; WC: well-classified.

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Fig S17.DS1 mean waveforms of ApoE3-KI and ApoE4-KI mice.(A) DS1 mean waveforms of each ApoE3-KI mouse (left panel) and each ApoE4-KI mouse (middle panel) when young.Numbers within the graphs indicate the average number of events with minimum and maximum values in parentheses.The comparison of overall mean waveforms is shown in the right panel.(B,C) Same as A, but for mice in adult and old ages.ApoE3-KI: Apolipoprotein E3 knock-in mice; ApoE4-KI: Apolipoprotein E4 knock-in mice; DS1: dentate spike type 1; DS2: dentate spike type 2.

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Fig S18.DS2 mean waveforms of ApoE3-KI and ApoE4-KI mice.(A) DS2 mean waveforms of each ApoE3-KI mouse (left panel) and each ApoE4-KI mouse (middle panel) when young.Numbers within the graphs indicate the average number of events with minimum and maximum values in parentheses.The comparison of overall mean waveforms is shown in the right panel.(B,C) Same as A, but for mice in adult and old ages.ApoE3-KI: Apolipoprotein E3 knock-in mice; ApoE4-KI: Apolipoprotein E4 knock-in mice; DS1: dentate spike type 1; DS2: dentate spike type 2.

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Fig S19.Stable DS metrics across genotypes, ages and datasets.(A) Comparison of the scaled peak amplitudes of DS1 (left panel) and DS2 (right panel) between young ApoE3-KI and ApoE4-KI mice.(B,C) Same as A, but for mice in adult and old ages.(D) Comparison of DS1 widths of ApoE3-KI (left panel) and ApoE4-KI mice (right panel) across ages.(E) Same as D, but for DS2.(F) Comparison of scaled peak amplitudes of DS1 (left panel) and DS2 (right panel) between mice from datasets A-C and ApoE transgenic mice, whose DSs were classified via CSDbC and WFbC respectively.(G) Same as F, but for DS widths.Statistics and p-values were obtained from Mann-Whitney U tests for panels in A-C,F,G, and from Kruskal-Wallis H tests for panels in D and E. ApoE3-KI: Apolipoprotein E3 knock-in mice; ApoE4-KI: Apolipoprotein E4 knock-in mice; DS1: dentate spike type 1; DS2: dentate spike type 2.